Expression Of Endothelial Growth Factor Receptor Research Articles

A therapeutically relevant, 3,3′-diindolylmethane derivative NGD16 attenuates angiogenesis by targeting glucose regulated protein, 78 kDa (GRP78)

Angiogenesis remain a critical procedure for tumor progression and malignancy. Anticancer agents targeting angiogenic cascades have been proved to be an effective strategy in the field of cancer therapeutics. The current study aims to explore the mechanistic prevention of angiogenesis and cancer cell proliferation by 1,1′-β-d-glucopyranosyl-3,3′-bis(5-bromoindolyl)-octyl methane (NGD16), a novel N-glycosylated derivative of 3,3′-diindolylmethane (DIM). NGD16 suppressed the viability of prostate cancer (PC-3), pancreatic adenocarcinoma (MiaPaca-2), colorectal cancer (COLO-205) and human umbilical vein endothelial cells (HUVECs) effectively with IC50 values 0.8μM, 2.8μM, 5.3μM and 2.5μM respectively. Abrogation of angiogenesis by NGD16 was promising in in vivo mouse Matrigel plug assay as well as in ex vivo sprouting of rat thoracic aorta. At the molecular level, NGD16 inhibited the expression of glucose regulated protein, 78kDa (GRP78), vascular endothelial growth factor receptor-2 (VEGFR2) and matrix metalloproteinase-9 (MMP-9) expression, the main mediators of angiogenesis and neovessel formation. Overexpression of GRP78 upregulated the expression of MMP-9 and VEGFR2 in PC-3 and HUVECs. Antibody blocking of GRP78 further potentiated NGD16 in attenuating angiogenesis through inhibition of MMP-9. NGD16 depicted its promising biodistribution profile in a pharmacokinetic study with 46.9% intraperitoneal bioavailability. Our findings suggest NGD16 is a potent inhibitor of neo-angiogenesis with a desirable pharmacokinetic profile, which can be taken forward in its development as an anticancer drug.

Antineoplastic effects of Chlorella pyrenoidosa in the breast cancer model

ObjectivesThere has been considerable interest in both clinical and preclinical research about the role of phytochemicals in the reduction of risk for cancer in humans. The aim of this study was to determine the antineoplastic effects of Chlorella pyrenoidosa in experimental breast cancer in vivo and in vitro. MethodsIn this experiment, the antineoplastic effects of C. pyrenoidosa in the chemoprevention of N-methyl-N-nitrosourea–induced mammary carcinogenesis in female rats were evaluated. Chlorella powder was administered through diet at concentrations of 0.3% and 3%. The experiment was terminated 14 wk after carcinogen administration. At autopsy, mammary tumors were removed and prepared for histopathological and immunohistochemical analysis. In vitro cytotoxicity assay, parameters of apoptosis, and proliferation after chlorella treatment in human breast adenocarcinoma (MCF-7) cells were carried out. ResultsBasic parameters of experimental carcinogenesis, mechanism of action (biomarkers of apoptosis, proliferation, and angiogenesis), chosen metabolic variables, and side effects after long-term chlorella treatment in animals were assessed. Chlorella at higher concentration suppressed tumor frequency by 61% (P < 0.02) and lengthened tumor latency by 12.5 d (P < 0.02) in comparison with the controls. Immunohistochemical analysis of rat tumor cells showed caspase-7 expression increase by 73.5% (P < 0.001) and vascular endothelial growth factor receptor-2 expression decrease by 19% (P = 0.07) after chlorella treatment. In a parallel in vitro study, chlorella significantly decreased survival of MCF-7 cells in a dose-dependent manner. In chlorella-treated MCF-7 cells, a significant increase in cells having sub-G0/G1 DNA content and significant increase of early apoptotic and late apoptotic/necrotic cells after annexin V/PI staining assay were found. Decreases in mitochondrial membrane potential and increasing reactive oxygen species generation were observed in the chlorella-treated MCF-7 cells. ConclusionsThis study is the first report on the antineoplastic effects of C. pyrenoidosa in experimental breast cancer in vivo and in vitro.

Systematic evaluation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance

Objectives: The identification of novel molecular biomarkers, predicting outcome of ovarian cancer, is highly desirable. Considering that angiogenesis is a critical factor for ascites development and peritoneal dissemination in ovarian cancer and given that the vascular endothelial growth factor (VEGF) receptor signaling axis is a major driver of angiogenesis, we sought to analyze expression and compartmental distribution of VEGF-receptor family in ovarian cancer and to assess its clinical relevance with regard to established clinicopathological parameters, tumor cell dissemination to the bone marrow (BM) and the patient's survival.

Differences in vascular endothelial growth factor receptor expression and correlation with the degree of enhancement in medulloblastoma.

Vascular endothelial growth factor (VEGF) is the major proangiogenic factor in many solid tumors. Vascular endothelial growth factor receptor (VEGFR) is expressed in abundance in pediatric patients with medulloblastoma and is associated with tumor metastasis, poor prognosis, and proliferation. Gadolinium enhancement on MRI has been suggested to have prognostic significance for some tumors. The association of VEGF/VEGFR and Gd enhancement in medulloblastoma has never been closely examined. The authors therefore sought to evaluate whether Gd-enhancing medulloblastomas have higher levels of VEGFR and CD31. Outcomes and survival in patients with enhancing and nonenhancing tumors were also compared. A retrospective analysis of patients with enhancing, nonenhancing, and partially enhancing medulloblastomas was performed. Primary end points included risk stratification, extent of resection, and perioperative complications. A cohort of 3 enhancing and 3 nonenhancing tumors was selected for VEGFR and CD31 analysis as well as microvessel density measurements. Fifty-eight patients were analyzed, and 20.7% of the medulloblastomas in these patients were nonenhancing. Enhancing medulloblastomas exhibited strong VEGFR1/2 and CD31 expression relative to nonenhancing tumors. There was no significant difference in perioperative complications or patient survival between the 2 groups. These results suggest that in patients with medulloblastoma the presence of enhancement on MRI may correlate with increased vascularity and angiogenesis, but does not correlate with worse patient prognosis in the short or long term.

Abstract 252: Is Vascular Disease a Result of Too Much Vascular Damage? Evidence for an Active Endogenous Plasma Membrane Repair System in Endothelial Cells

Many studies have correlated vascular disease with excessive vascular damage. For instance, vascular endothelial cells (EC) at sites of arterial branching are exposed to turbulent flow, a highly atherogenic factor, whereas chronic hypertension accelerates endothelial dysfunction. At the molecular level, EC are routinely exposed to damaging oxidants, cholesterol and cigarette smoke chemicals. However, how the vascular endothelium ‘repairs’ after ‘damage’ was until recently unknown. Recently, we have shown that EC express FERLINS ‘repair’ proteins, documented to allow the fusion of lipid ‘patches’ to a damaged plasma membrane. Indeed, Dysferlin and Myoferlin mRNA and proteins were found to be highly expressed in every EC line and human/rodent blood vessel investigated. Proteomics analyses revealed that both Dysferlin and Myoferlin are caveolae/lipid raft resident proteins, well known to be rich in signaling proteins at the heart of laminar flow sensing/ mechanotransduction. In vitro, loss of Dysferlin and Myoferlin resulted in attenuated Platelet Endothelial Cellular Adhesion Molecule-1 (PECAM-1) and Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) expression, respectively, as well as blunted membrane repair following physical injury. In vivo, Dysferlin-null and Myoferlin-null mice show blunted angiogenic response in response to various stimuli. Together, these data clearly show that the vasculature has complex, endogenous physical repair machinery at the plasmalemma, that the lipid ‘patches’ likely contain transmembrane protein cargo, and that Ferlins clearly play a role in vascular homeostasis. Finally, recent preliminary data from our laboratory suggests that Dysferlin is highly overexpressed in human atherosclerotic sections, and that Dysferlin-null mice are more susceptible to cardiovascular disease that their WT counterparts, indicating a protective role for Ferlin-associated repair in the cardiovascular system.

Explorative investigation of vascular endothelial growth factor receptor expression in primary ovarian cancer and its clinical relevance

ObjectivesThe identification of novel molecular biomarkers, predicting outcome of ovarian cancer, is highly desirable. Considering that angiogenesis is a critical factor for ascites development and peritoneal dissemination in ovarian cancer and given that the vascular endothelial growth factor (VEGF) receptor signaling axis is a major driver of angiogenesis, we sought to analyze expression and compartmental distribution of VEGF-receptor family in ovarian cancer and to assess its clinical relevance with regard to established clinicopathological parameters, tumor cell dissemination to the bone marrow (BM) and the patient's survival. MethodsA total of 73 patients with primary ovarian cancer were enrolled into this study. Primary tumor tissue was analyzed for the expression of VEGF-R1, VEGF-R2 and VEGF-R3 by immunohistochemistry. The presence of disseminated tumor cells (DTC) in the BM was analyzed by immunocytochemistry using the pancytokeratin antibody A45B/B3 and subsequent automatic detection based on staining and cytomorphology. ResultsIn primary ovarian cancer tissue, VEGF-receptor expression, detected with an overall frequency of 44%, was mostly located in the vascular wall and across the stroma; positivity rates for VEGF-R1, VEGF-R2 and VEGF-R3 were 34%, 18% and 26%, respectively. Total VEGF-receptor expression correlated with residual tumor after primary debulking surgery and the presence of DTC at primary diagnosis (p=0.035, p=0.023, respectively). Interestingly, VEGF-R1 positivity significantly correlated with decreased progression-free survival (p=0.026). ConclusionsThis is the first report, suggesting total VEGF-receptor status as a molecular biomarker for monitoring tumor cell spread to the BM and, particularly, revealing prognostic significance of VEGF-R1.

Comparison of the biological characteristics of early and late endothelial progenitor cells derived from mouse bone marrow

Objective To observe the biological characteristics and precise role in angiogenesis of endothelial progenitor cells (EPCs).Methods Bone marrow-derived mononuclear cells were isolated and cultured.EPCs were identified by using immunohistochemistry,immunofluorescence and confocal microscopy.Using methyl thiazol tetrazolium(MTT) method,three-dimensional culture and flow cytometry,the characteristics of early and late EPCs were compared.The features of EPCs were observed by transmission electron microscopy.Results EPCs expressed cell surface antigen positive markers.After two weeks the late EPCs had greater proliferation,achieving 1 × 1011/L,and could form vascular-like structure.Leukocyte differentiation antigen 133 (CD133) expression of the early EPCs was up to (88.7 ± 2.4)％,then late EPCs was gradually decreased to (46.4 ± 3.6) ％.But vascular endothelial growth factor receptor-2 (VEGFR-2) expression was increased from (55.3 ±2.5)％ up to (81.1 ± 1.6)％,and there was significant difference between early EPCs and late EPCs (P ＜ 0.05).The late EPCs could be seen in the endothelial cells characteristic cell-Weible-Palade (WP) bodies.Conclusion There are different qualities of early EPCs and late EPCs in the bone marrow.They show some of the same phenotypes but there are also differences.The late EPCs may be an attractive candidate cell for angiogenesis. Key words: Bone marrow; Endothelial progenitor cells ; Endothelial cells ; Angiogenesis

Preventive effects of fluvastatin in rat mammary carcinogenesis

On the basis of preclinical and clinical evidence, statins lead to risk reduction of several types of neoplasia including breast cancer. This study is the first report on the preventive effects of fluvastatin in experimental breast cancer in vivo. In this experiment, the antineoplastic effects of fluvastatin in the chemoprevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats were evaluated. The effects of fluvastatin on selected parameters of apoptosis, proliferation, and angiogenesis in mammary tumor cells were determined. The drug was dietary administered at two concentrations of 20 and 200 mg/kg. The experiment was terminated 17 weeks after carcinogen administration; mammary tumors were removed and prepared for histomorphological and immunohistochemical analysis. The basic parameters of experimental carcinogenesis, chosen metabolic variables, and side effects after long-term fluvastatin treatment in animals were assessed. Fluvastatin at higher concentrations suppressed tumor frequency by 63% and tumor incidence by 33% in comparison with the controls. After fluvastatin treatment, immunohistochemical analysis of tumor cells showed a decrease in vascular endothelial growth factor receptor-2 expression by 86% and an increase in caspase-3 by 8.5%. Fluvastatin in both treated groups significantly increased the parameters of serum lipid metabolism and significantly decreased femur compact bone thickness and body weight in animals. Our results suggest that fluvastatin and other statins should be further evaluated for tumor-preventive characteristics.

Vascular Endothelial Growth Factor Receptor-1 Expression in Breast Cancer and Its Correlation to Vascular Endothelial Growth Factor A

VEGF-A is the most potent angiogenic factor in tumour angiogenesis. Its effects are mediated via two receptors VEGFR-1 and VEGFR-2. Primary aim of our study was to examine the expression of VEGFR-1 in breast cancer and its correlation to VEGF expression, lymph node status, tumour size, histological grade, and hormone receptor status. To examine the VEGFR-1 and VEGF expressions in tumour and surrounding tissue of 51 breast cancer patients, and in healthy breast tissue of 30 benign breast diseases patients, we used three-step immunohistochemical staining. VEGFR-1 and VEGF expressions were significantly increased in breast cancer tumour in relation to surrounding tissue (P < 0.01), and the VEGF expression was significantly increased in lymph node positive breast cancer patients (P < 0.01). VEGFR-1 and VEGF expressions were significantly higher in breast cancer tumour compared with healthy breast tissue (P < 0.01). Significant correlation between VEGF and VEGFR-1 expressions was found (P < 0.05). No significant correlations between VEGF and VEGFR-1 expressions and tumour size, histological grade, and hormone receptor status were found. Increased expression of VEGFR-1 and VEGF in breast cancer tumour and significant correlation between these proteins suggest the possible role of VEGF/VEGFR-1 signalization in breast cancer development, although VEGFR-1 potential prognostic value was not confirmed.

MicroCT based angiography for studying neovascularization of long bone fracture repair in a rat model

Objective To explore microcomputed tomography (MicroCT) based angiography for exhibiting the neovascularization/angiogenesis of a callus on a rat femoral fracture model.Methods After a closed fracture establishment,60 nale SD rats were randomized into2 groups (30 in each group)and killed at the time point of 1,2,3,4 and 8 weeks The callus in experimental group were scanned by MieroCT and 3-D vasculature images were reconstructed.Vessel size distribution,total vessel volume and volume fraction were quantified.The callus in control group were assessed by using immunohistochemisty for observing the expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 ( VEGFR-2 ).Results MicroCT based angiography provided native 3-D vasculature images to iconicly reveal the states of neovascularization.And the total vessel volume and volume fraction peaked at 3 weeks ( P ＜ 0.05 ):( 196.00 ± 20.33 ) mm3 and ( 6.70 ± 0.74 ) ％ respectively ; Immunohistochemistry of callus sections showed the expression of VEGF and VEGFR-2 occurred in the early stage of fracture healing and peaked at 2 weeks,the number of positive cells were ( 113.40 ± 9.17 ) and ( 51.80 ± 4.24 ) respectively ( P ＜0.05).Conclusion MicroCT based angiography atraumaticly provided high-resolution,quantitative,3-dimention,and objective data analysis.MicroCT based angiography is a robust methodology for evaluation of vascular networks in the callus of a small animal. Key words: MicroCT; Perfusion; Angiogenesis; Fracture; Immunohistochemistry

Systemic anti-vascular endothelial growth factor therapies induce a painful sensory neuropathy

Systemic vascular endothelial growth factor inhibition, in combination with chemotherapy, improves the outcome of patients with metastatic cancer. Peripheral sensory neuropathies occurring in patients receiving both drugs are attributed to the chemotherapy. Here, we provide unprecedented evidence that vascular endothelial growth factor receptor inhibitors trigger a painful neuropathy and aggravate paclitaxel-induced neuropathies in mice. By using transgenic mice with altered neuronal vascular endothelial growth factor receptor expression, systemic inhibition of vascular endothelial growth factor receptors was shown to interfere with the endogenous neuroprotective activities of vascular endothelial growth factor on sensory neurons. In vitro, vascular endothelial growth factor prevented primary dorsal root ganglion cultures from paclitaxel-induced neuronal stress and cell death by counteracting mitochondrial membrane potential decreases and normalizing hyperacetylation of α-tubulin. In contrast, vascular endothelial growth factor receptor inhibitors exerted opposite effects. Intriguingly, vascular endothelial growth factor or vascular endothelial growth factor receptor inhibitors exerted their effects through a mechanism whereby Hdac6, through Hsp90, controls vascular endothelial growth factor receptor-2-mediated expression of the anti-apoptotic Bcl2. Our observations that systemic anti-vascular endothelial growth factor therapies interfere with the neuroprotective activities of vascular endothelial growth factor may have important implications for the application of anti-vascular endothelial growth factor therapies in cancer patients.

Sunitinib Malate Provides Activity Against Murine Bladder Tumor Growth and Invasion in a Preclinical Orthotopic Model

To evaluate the effects of sunitinib on localized bladder cancer in a mouse orthotopic bladder tumor model. We used an established orthotopic mouse bladder cancer model in syngeneic C3H/He mice. Treatment doses of 40 mg/kg of sunitinib or placebo sterile saline were administrated daily by oral gavage. Tumor volume, intratumoral perfusion, and in vivo vascular endothelial growth factor receptor-2 expression were measured using a targeted contrast-enhanced micro-ultrasound imaging system. The findings were correlated with the total bladder weight, tumor stage, and survival. The effects of sunitinib malate on angiogenesis and cellular proliferation were measured by immunostaining of CD31 and Ki-67. Significant inhibition of tumor growth was seen after sunitinib treatment compared with the control. The incidence of extravesical extension of the bladder tumor and hydroureter in the sunitinib-treated group (30% and 20%, respectively) was lower than the incidence in the control group (66.7% and 55.6%, respectively). Sunitinib therapy prolonged the survival in mice, with statistical significance (log-rank test, P = .03). On targeted contrast-enhanced micro-ultrasound imaging, in vivo vascular endothelial growth factor receptor-2 expression was reduced in the sunitinib group and correlated with a decrease in microvessel density. The results of our study have demonstrated the antitumor effects of sunitinib in the mouse localized bladder cancer model. Sunitinib inhibited the growth of bladder tumors and prolonged survival. Given that almost 30% of cases in our treatment arm developed extravesical disease, sunitinib might be suited as a part of a multimodal treatment regimen for bladder cancer.

Gremlin-1 Induces BMP-Independent Tumor Cell Proliferation, Migration, and Invasion

Gremlin-1, a bone morphogenetic protein (BMP) antagonist, is overexpressed in various cancerous tissues but its role in carcinogenesis has not been established. Here, we report that gremlin-1 binds various cancer cell lines and this interaction is inhibited by our newly developed gremlin-1 antibody, GRE1. Gremlin-1 binding to cancer cells was unaffected by the presence of BMP-2, BMP-4, and BMP-7. In addition, the binding was independent of vascular endothelial growth factor receptor-2 (VEGFR2) expression on the cell surface. Addition of gremlin-1 to A549 cells induced a fibroblast-like morphology and decreased E-cadherin expression. In a scratch wound healing assay, A549 cells incubated with gremlin-1 or transfected with gremlin-1 showed increased migration, which was inhibited in the presence of the GRE1 antibody. Gremlin-1 transfected A549 cells also exhibited increased invasiveness as well as an increased growth rate. These effects were also inhibited by the addition of the GRE1 antibody. In conclusion, this study demonstrates that gremlin-1 directly interacts with cancer cells in a BMP- and VEGFR2-independent manner and can induce cell migration, invasion, and proliferation.

Etiology of OHSS and use of dopamine agonists

Vascular endothelial growth factor is the factor that causes increased capillary permeability and therefore the most serious manifestations of ovarian hyperstimulation syndrome. Dopamine agonists can both prevent and treat ovarian hyperstimulation syndrome by blocking expression of the vascular endothelial growth factor receptor.

Aldosterone inhibits endothelial morphogenesis and angiogenesis through the downregulation of vascular endothelial growth factor receptor-2 expression subsequent to peroxisome proliferator-activated receptor gamma

Angiogenesis plays a pivotal role in cardiovascular diseases such as ischemic heart disease, limb ischemia and heart failure, and has recently been shown to mediate various biological activities related to the pathogenesis of these diseases. In the present study, we evaluated the role of aldosterone in angiogenesis. Tube formation assay on Matrigel using human umbilical vein endothelial cells (HUVEC) revealed that aldosterone inhibited endothelial morphogenesis in a manner sensitive to eplerenone, a selective mineralocorticoid receptor antagonist. The anti-angiogenic effect of aldosterone was further confirmed by an in vivo angiogenesis assay using a Matrigel plug model in mice. Reverse transcription-mediated polymerase chain reaction and immunoblotting demonstrated that aldosterone downregulated the expression levels of vascular endothelial growth factor receptor-2 (VEGFR-2) and peroxisome proliferators-activated receptor gamma (PPAR gamma). VEGFR-2 expression was found to be enhanced in response to PPAR gamma activation by troglitazone, and attenuated by GW9662, a specific antagonist of PPAR gamma. In the tube formation assay, endothelial morphogenesis was stimulated by troglitazone, and inhibited by GW9662, indicating that PPAR gamma activation mediates positive regulation of angiogenesis through enhancement of VEGFR-2 expression. These data suggest that aldosterone inhibits angiogenesis through VEGFR-2 downregulation, subsequent to, at least in part, attenuation of PPAR gamma expression. The present findings provide a new insight into the possible therapeutic application of mineralocorticoid receptor blockade to various cardiovascular diseases.

Chronic Deficiency of Nitric Oxide Affects Hypoxia Inducible Factor-1α (HIF-1α) Stability and Migration in Human Endothelial Cells

BackgroundEndothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated.ResultsEndothelial nitric oxide synthase (eNOS) was chronically inhibited either by N G-Nitro-l-arginine methyl ester (l-NAME) treatment or its expression was down-regulated by RNA interference. After long-term l-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O2 consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells.ConclusionBased on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.

Uterine Glomeruloid Hemangioma in a Patient Without POEMS Syndrome

Cutaneous glomeruloid hemangioma is a hallmark of POEMS syndrome. These patients have elevated serum levels of vascular endothelial growth factor. The authors report an extracutaneous uterine glomeruloid hemangioma in an 82-year-old woman with a history of breast and endometrial carcinomas. Within the lumen of myometrial vessels, a lobular, glomeruloid proliferation of capillary-like CD31 and vascular endothelial growth factor receptor-1-positive endothelial cells was found. The capillary loops were lined by endothelial cells, most of them containing PAS-positive and immunoglobulin-positive eosinophilic hyaline globules (thanatosomes). This vascular proliferation was consistent with a glomeruloid hemangioma. Although an extracutaneous glomeruloid vascular proliferation has been found in the retroperitoneal adipose tissue in a patient with POEMS syndrome, this study reports what seems to be the first case of visceral glomeruloid hemangioma in a patient without POEMS syndrome. The authors hypothesize that the glomeruloid endothelial cell proliferation with vascular endothelial growth factor receptor-1 expression may be a paraneoplastic phenomenon.

Correlation of Immunohistologic and Perfusion Vascular Parameters with MR Contrast Enhancement Using Image-guided Biopsy Specimens in Gliomas

The purpose of this study was to correlate the status of magnetic resonance contrast enhancement with immunohistologic vascular parameters such as microvascular cellular proliferation (MVCP), microvascular density (MVD), vascular endothelial growth factor receptor-2 (VEGFR-2) expression, and World Health Organization (WHO) grade obtained from image-guided biopsy specimens. We also compared perfusion computed tomography (PCT) parameters such as cerebral blood volume (CBV), cerebral blood flow (CBF), and permeability surface area-product (PS) with the presence or absence of contrast enhancement. A total of 26 image-guided biopsy specimens in 16 patients with treatment naive gliomas were obtained from contrast-enhancing (CE) and nonenhancing (NE) regions of the glioma. Contrast enhancement status was correlated with MVD, MVCP, VEGFR-2 expression, and WHO grade obtained from the biopsy specimen as well as with the PCT parameters. Contrast enhancement showed statistically significant correlation with MVCP (P = .003) and PS (P = .007) when compared with various immunohistologic and perfusion vascular parameters. WHO grade of the biopsy specimen showed statistically significant correlation with contrast enhancement (P = .002), MVCP (P < .001), and PS values (P = .028). Contrast enhancement in gliomas is primarily from a break in blood-brain barrier as evidenced by its correlation with PS and MVCP, whereas it was not statistically correlated with CBV and MVD even though it showed a positive trend. Contrast enhancement also showed significant correlation with WHO grade suggesting a biopsy from CE region in a heterogeneous glioma probably will still yield the most aggressive part of the glioma is also shown by its association with MVCP and PS estimates.

Biomarkers for Cetuximab-Based Neoadjuvant Radiochemotherapy in Locally Advanced Rectal Cancer

Phase II trials in locally advanced rectal cancer have shown that cetuximab-based neoadjuvant radiochemotherapy is feasible but without an improvement in complete pathologic response rates. Our goal was to identify patients who would benefit from cetuximab-based neoadjuvant chemoradiation measuring gene expression levels of proteins involved in tumor growth [endothelial growth factor receptor (EGFR)], angiogenesis [VEGF, VEGF receptors 1 and 2 (VEGFR1, VEGFR2)], DNA repair [excision repair cross-complementing 1 (ERCC1)], and drug metabolism [thymidylate synthetase (TS)]. We also determined mutation status of KRAS and BRAF. This study was carried out on 130 patients with locally advanced rectal cancer who were enrolled in 4 different phase II clinical trials, using cetuximab-based chemoradiation. Tumor tissues were obtained before neoadjuvant and at surgical therapy. After microdissection, intratumoral gene expression levels and KRAS/BRAF mutation status were analyzed. A significant decrease of TS, VEGFR1, and VEGFR2 gene expression was seen following neoadjuvant therapy (P < 0.03). High pretreatment VEGF gene expression levels were associated with nonresponse (P = 0.070). KRAS mutations were found in 42% and mutant KRAS (KRAS mt) was significantly associated with pathologic nonresponse (P = 0.037). In patients with wild-type KRAS (KRAS wt), low EGFR was significantly associated with higher nonresponse and VEGF mRNA expressions were associated with complete pathologic response (P = 0.012; P = 0.06). KRAS transversion (KRAS tv) was associated with tumor regression: nonresponse was more common in patients with KRAS tv than with KRAS wt (P = 0.007). BRAF V600E mutations were not detected in any of the patients. This study suggests that pretreatment intratumoral EGFR and VEGF mRNA expression levels as well as KRAS mutation status are predictive markers of pathologic response to neoadjuvant cetuximab-based chemoradiation in locally advanced rectal cancer.

Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo

Angiogenesis consists in the growth of new blood vessels from pre-existing ones. Although anti-angiogenesis interventions have been shown to have therapeutic properties in human diseases such as cancer, their effect is only partial and the identification of novel modulators of angiogenesis is warranted. Recently, we reported the unexpected proteomic identification in endothelial cells (EC) of Myoferlin, a member of the Ferlin family of transmembrane proteins. Ferlins are well known to regulate the fusion of lipid vesicles at the plasma membrane in muscle cells, and we showed that Myoferlin gene knockdown not only decreases lipid vesicle fusion in EC but also attenuates Vascular Endothelial Growth Factor (VEGF) Receptor-2 (VEGFR-2) expression. Herein, we show that Myoferlin gene silencing in cultured EC also results in attenuated expression of a second tyrosine kinase receptor, Tie-2, which is another well-described angiogenic receptor. Most importantly, we provide evidence that delivery of a low-volume Myoferlin siRNA preparation in mouse tissues results in attenuated angiogenesis and edema formation. This provides the first evidence that acute Myoferlin knockdown has anti-angiogenic effects and validates Myoferlin as an anti-angiogenesis target. Furthermore, this supports the unexpected but increasingly accepted concept that proper tyrosine kinase receptors expression at the plasma membrane requires Myoferlin.